Troubleshooting

General Recommendations

Store reagents properlyRead the data sheet carefully and store reagents in their proper location. Some kits require different components to be stored at different temperatures.

Equilibrate reagents to assay temperatureUse an incubator or water bath to equilibrate all reagents (except enzymes) to assay temperature. Enzymes should be thawed and kept on ice or at 4°C during assays.

Run a test standard curveMake sure that there is nothing faulty with the reagents and to ensure that you are following the directions carefully. Your standard curve should be linear unless otherwise noted in data sheet and generate similar results to the example graph.

Preliminary sample serial dilutionIf this is your first time running a particular type of sample, use micro-centrifuge tubes to make a 2x serial dilution of your sample. Run the assay with the original and diluted samples to determine if dilution is necessary and which dilution factor is optimal. Use the chosen dilution factor in all subsequent experiments.

Pipette carefullyConsistency between wells is very important; carefully pipette down the side of the well and ensure that all wells have the same volume and all reagents flow down to the bottom. Take special care to avoid bubbles, which can disrupt absorbance readings.



Troubleshooting

Assay Not Working (No Signal)

POSSIBLE CAUSESSOLUTIONS
Assay buffer is too cold, causing low enzyme activity

Equilibrate reagents to room temperature or specified assay temperature.

Omission of a reagent or step in the protocolRe-read the data sheet and follow instructions carefully.

Plate read at incorrect wavelengthRe-read the data sheet and follow instructions carefully.

Use of an incorrect microplateAbsorbance: clear plates;
Fluorescence: black plates (bottoms can be clear or black);
Luminescence: white plates.

POSSIBLE CAUSESSOLUTIONS
Incompatible sample typeRe-read the data sheet for list of compatible samples.

Improper sample preparationFor enzyme activity assays, do not deproteinate your sample. Deproteination kills all enzyme activity. If you do need to deproteinate, make sure that you have neutralized your sample properly. EDTA is a metal chelator, and interferes with a few of our assays. Any assay that measures metal ions or requires a metal cofactor is incompatible with EDTA.

POSSIBLE CAUSESSOLUTIONS
Standards were made too concentrated, saturating the signalRefer back to the data sheet and ensure that standard dilutions are done correctly.

Working reagent was made incorrectly, saturating the signalRefer back to the data sheet and ensure that working reagent was made correctly.

Samples are too concentratedDilute your samples and repeat the experiment.

POSSIBLE CAUSESSOLUTIONS
Reagents incorrectly storedCheck that reagents have been properly stored. They may degrade rapidly if incorrectly stored.

Reagents have expiredCheck the shelf life of the kit.

Samples are too diluteConcentrate or remake your samples with more cells/tissue.

POSSIBLE CAUSESSOLUTIONS
Unmixed and non-uniform wellsTap plate a few times quickly to mix contents thoroughly.

Bubbles in the wellsCheck wells for air bubbles. Repeat assay and pipette carefully avoiding bubbles.

Precipitate in the wellsCheck well for precipitates or turbidity. Determine cause of precipitation and dilute, deproteinate, or use some other alternative sample treatment to eliminate precipitation.

POSSIBLE CAUSESSOLUTIONS
Pipetting errors and variabilityRemake standard dilutions and closely follow instructions while carefully pipetting.

Incorrect calculationsRe-check the calculations. Some assays have different equations.

Standard curve maybe a non-linear fitRefer to the data sheet for the fitting equation and example graph.



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